Many factors regulate normal cerebral blood flow (CBF), including signaling molecules that are synthesized and released from cells in the vicinity of cerebral blood vessels. Pathological changes in the brain also affect CBF; for example, CNS inflammation can be accompanied by abnormalities in CBF that lead to life-threatening edema. The hypothesis to be tested by the experiments in this proposal is that endocannabinoids are synthesized by both perivascular microglia (resident immune cells of the brain) and cerebral endothelial cells and m CBF under both normal and pathological conditions. In addition, we hypothesize that endocannabinoids produce vasodilation via vascular smooth muscle (VSM) CB1 cannabinoid receptor activation. Support for this hypothesis includes our findings that: the CB1 cannabinoid receptor is expressed by cerebral arterial and arteriolar VSM cells; activation of the VSM CB1 receptorand results in inhibition of L-type calcium channels; cannabinoids vasodilate isolated cerebral vessels and increase CBF in vivo. Intact cerebral arteries synthesize endocannabinoids in response to microglial and endothelial activators, supporting the hypothesis that cells in the immediate vicinity of the VSM make endocannabinoids. In addition, microglial cells synthesize endocannabinoids in response to an activating stimulus, lipopolysaccharide (LPS). The primary model that we will use to approach this hypothesis is the isolated, middle cerebral artery from rat. We will also use atmospheric liquid chromatography-mass spectrometry to measure endocannabinoids and the in vivo significance of these findings will be explored using laser Doppler flowmetry. The specific aims of this proposal are: (1) to characterize the role of the CB1 receptor in cerebral arterial vasomotor tone and responsiveness; (2) to determine the distribution of the CB1 receptor throughout the cerebral vasculature; (3) to determine the cellular sources and regulation of endocannabinoid production in isolated cerebral arteries; (4) to determine whether activated microglial cells induce CB1 receptor-dependent vasodilation cerebral arteries; and (5) to determine the contribution of VSM CB1 receptors in the regulation of cerebral blood flow in vivo. Activated microglial cells contribute to many neurodegenerative diseases and to the worsening of stroke and trauma. Increased understanding of the mediators that are released by activated microglia and their targets may provide new therapeutic approaches to CNS diseases with an inflammatory component.